The goal of this application is to investigate the important role of intestinal microbial homeostasis in ALS, meanwhile investigating a novel approach that can potentially treat ALS by restoring host-microbe relationships and combining with FDA approved drugs. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by progressive death of motor neurons and muscle atrophy. Most patients die within 5 years after the disease onset. Currently, treatment with Riluzole and Radicava only extends patient life span for a few months. Therefore, there are significant needs to develop novel treatments for ALS and improving the life quality of ALS patients. Emerging evidence has demonstrated that microbiome and intestinal homeostasis plays essential roles in neurological diseases, such as Alzheimer's disease and Parkinson's disease. However, little is known about the intestinal microbiome in patients with ALS. Our lab is the first to discover the link between intestinal homeostasis and the disease progression in an ALS mouse model G93A. Our study in human ALS further reveals the dysbiosis and intestinal inflammation. ALS mice had damaged intestinal structure and increased intestinal permeability (leaky gut). Remarkably, restoring the intestinal homeostasis by feeding the ALS model mice with a bacterial product butyrate significantly delayed the disease onset and prolonged the life span of ALS mice. We hypothesize that targeting gut microbiome and combining with FDA approved drugs to improve the intestinal barrier function and restore microbiome, thus slowing disease progression of ALS. The studies are designed to rigorously examine the hypotheses and address two main objectives. Aim 1 is to determine the mechanisms that contribute to dysbiosis and barrier dysfunction in ALS. We will define the mechanism for abnormal epithelial junction structure and dysbiosis (e.g. loss of butyrate- producing bacteria) in ALS mice. We will investigate roles of butyrate and butyrate-producing bacteria, using novel molecular tools, enteroid cultures, and transgenic animal models (SOD1G93 mice and a novel gut-specific overexpression of SOD1G93 model). We will determine mechanisms underlying the benefits of bacterial product butyrate on intestinal permeability and neuroprotection. Aim 2 is to conduct proof-of-principle studies with restoring gut microbiome and intestinal barrier for preserving neuromuscular function in ALS. ALS mice at different stages of ALS will be treated with beneficial bacteria (probiotics that promote butyrate production) or combining with FDA drugs (Riluzole or Radicava). ALS mice will have fecal microbiota transplantation (FMT) using health wild-type mice as donors. Physiological and molecular biological assessments of intestinal integrity, microbiome, and neuromuscular performance will be used to evaluate the therapeutic effects. Histopathology and biochemical evaluations will be used to determine changes at the cellular level of intestinal and neuromuscular function at different stages of ALS. We will optimize the efficacy of restoring intestinal microbiome combining with FDA drugs in slowing ALS progression using different ALS mouse models (SOD1G93A and TDP43). Veterans are twice as likely to be diagnosed with ALS as the general population. Military veterans, regardless of the branch of service, the era in which they served, or whether they served during a time of peace or war, are at a greater risk of dying from ALS than if they had not served in the military. Our study is significant because of the health burden of ALS in VA population and the novel role of the microbiome on neuromuscular function in health and disease, especially in Veterans' care. Innovation of this project lies in its: (a) therapeutic potential for ALS, (b) conceptual frame-work to discover early changes and dysbiosis and the gut barrier effects before onset of ALS, and (c) state-of?the-art experimental models that allow us to understand novel mechanisms underlying the beneficial effect of probiotics and/or bacterial products that improve the motor neuron function. It has significant translational implications for developing new therapeutic strategies for combating this devastating disease and improving the health of veterans.